Interferon gamma (IFN?) is essential for cell-autonomous resistance to an array of microbial pathogens. In the past decade, significant advances have been made in identifying and characterizing many of the IFN?-induced antimicrobial mechanisms that limit pathogen growth within host cells. These processes often result in direct killing of the pathogen, disruption of the pathogen's replicative niche, and/or sequestration of metabolites required for bacterial growth. Most of the cell-autonomous resistance mechanisms that have been described are targeted to microbes that replicate in pathogen containing vacuoles (PCVs). However, very little is known about how IFN? restricts the growth of bacteria, such as Shigella flexneri, that replicate in the host cytoplasm. S. flexner is a Gram-negative intracellular pathogen responsible for serious enteric infections, characterized by severe inflammatory bacillary dysentery. Type III-secreted effector proteins enable S. flexneri to establish a successful infectious cycle in which the bacteria invade nonphagocytic cells, lyse the resulting vacuole, and replicate in the host cell cytoplasm. In IFN?-activated cells, however, these bacterial effectors fail to overcome the host's defense system, shifting the balance in favor of the host and resulting in clearance of the bacteria. In our first aim, we will work to identify IFN?-dependent host gene products and/or pathways that restrict S. flexneri replication. As we began to investigate known IFN?-mediated effector mechanisms for their role in restricting S. flexneri, we discovered that the IFN?-inducible transcription factor interferon regulatory factor 1 (IRF1) is critical for S. flexneri growth restriction. This finding strongly suggests that target genes of IRF1 are critical for inhibiting S. flexneri growth. Therefoe in our first aim, we will first use microarrays to identify IFN?- dependent genes that are dependent on IRF1 for their transcription. We will then knock down each of these genes using lentivirus-delivered shRNA to identify host resistance genes that block S. flexneri replication. In our second aim, we will use straightforward experimental approaches to identify the step or steps of the S. flexneri developmental cycle (e.g. escape from the phagosome, intracellular spreading, survival in the cytosol) that are inhibited by IFN? during infection. Once we have identified a host gene product from Aim 1 that is involved in blocking S. flexneri replication, we will be able to explore more precisely how this mechanism(s) limits the progression of the infection within host cells. It is likely that the mechanisms that target cytosolic pathogens, or te pathways that lead to their activation, are different from those targeting pathogens that replicate in vacuoles. Only by understanding how IFN? constrains growth of cytosolic bacteria can we fully appreciate how this critical element of innate immunity might be better directed to control disease. PUBLIC HEALTH RELEVANCE: Shigella flexneri is a highly infectious bacterial pathogen that replicates inside intestines of humans causing dysentery. It is able to replicate inside intestinal cells, but little is known about mechanisms by which host cells ultimately slow down and stop the replication of these intracellular bacteria. Our goal is to identify the specific mechanisms used by the host cell to constrain infection with S. flexneri. Ultimately we may be able to strengthen immunity to this and other pathogens that use similar strategies to grow within human cells.